A caged molecule is an inert but photosensitive molecule that has latent biological activity. Light absorption transforms a caged molecule into a fully bioactive molecule. The photochemically generated molecules can be agonists or antagonists; therefore, photolysis of caged molecules is a convenient method for using light to switch biological processes on or off. Caged molecules are most often made by chemically modifying the structure of a bioactive molecule to mask its activity. Absorption of light then leads to photochemical alteration and/or cleavage of the activity-blocking modification to restore the activity of the biomolecule. Metaphorically, the biological activity of the molecule is "caged," and light is said to "uncage" or "photorelease" that activity.
Photolysis of caged molecules offers three advantages over conventional techniques of reagent delivery: temporal resolution, spatial resolution, and control over the chemical identity of the reagent being delivered. First, because they are biologically inactive, caged molecules can be preloaded into cells or preequilibrated with tissues before experimentation. A flash of light rapidly generates bioactive molecules in situ, which can immediately interact with their receptors. Problems with mixing, diffusion, or access to anatomically complex tissue are thus minimized. Photorelease typically occurs within a few microseconds to 1 ms, so neurophysiology can be manipulated with comparable time resolution. Second, a light beam is easy to manipulate and steer and can be focused to a diffraction-limited spot with diameter d = 1.22/NA, where is the wavelength of light used, and NA is the numerical aperture of the focusing lens. In typical experiments, photolysis is achieved by focusing UV light near 350 nm through a microscope objective with NA of >1.0, which means spatial resolution better than 0.5 µm is possible. Third, many biochemical messengers are chemically or enzymatically labile. Two examples are dopamine and anandamide; the former air-oxidizes readily, and the latter is cleaved rapidly by endogenous lipases. That the degradation products themselves can have independent biological action is problematic. Judicious caging converts labile molecules to stable species that resist chemical and biological breakdown. Focal photolysis then generates the authentic bioactive molecule in situ, thus affording precise control over the chemical identity of the applied reagent. Below, we describe novel caged compounds and technologies that illustrate the advantages of focal photolysis.